In 1998, approximately 29,000 people in the United States were diagnosed with pancreatic adenocarcinoma, and approximately 28,900 people were expected to die from this tumor [1]. The overall cure rate for pancreatic cancer remains less than 5% despite more than 20 years of clinical trials. Only 10% of subjects have a potentially resectable tumor; however, even for subjects undergoing a curative pancreaticoduodenectomy, five-year survival is 6-24% [2]. The vast majority of subjects have unresectable tumors and develop metastatic disease within the first year of therapy. The median survival for subjects with metastatic disease is 3-6 months.
The description of cancertrophic agents set forth below includes both growth factor and growth factor receptors which are surprisingly expressed or overexpressed in cancerous tumors or more specifically cancer cells.
Gastrin is highly expressed in the antral mucosa and duodenal bulb and expressed at low levels in a variety of tissues, including the pancreas. Gastrin is also highly expressed in the fetal pancreas, a fact which may be of significance in the development of pancreatic neoplasms [3].
The normal nonfetal pancreas shows no expression of gastrin isoforms or receptor. It has been shown that a large percentage of patients with pancreatic cancer possess progastrin, glycine-extended gastrin, amidated gastrin in their blood, and CCK-B/gastrin receptor are present in the tumor cells [4]. Thus, it was later found that pancreatic adenocarcinoma expresses the precursor forms of gastrin, especially the progastrin and glycine-extended forms. The tumor cells were also determined to express the CCK-B/gastrin receptor. Similarly, these precursor gastrin forms and receptors were detected in other cancers, such as gastric, colonic, and hepatocellular carcinomas.
Several growth factors have been postulated to affect the growth and development of pancreatic cancer. Moreover, it is well recognized that gastrin is a trophic hormone and promotes growth of gastrointestinal (G1) and non-gastrointestinal cancers [4]. Gastrin has been shown to promote the growth of hepatocellular carcinoma, renal cell carcinoma, small cell carcinoma of the lung and also pancreatic carcinoma [6-9]. Gastrin affects cell behavior in the form of circulating fully processed peptides as well as autocrine process whereby incomplete processed precursor gastrin, especially in the form of glycine-extended gastrin can stimulate cell growth or cell function [10].
In particular, a number of investigations have shown the important role that G17 gastrin and glycine-extended G17 (Gly-G17) gastrin play in the proliferation of gastrointestinal adenocarcinomas including pancreatic adenocarcinoma. It has been demonstrated that G17 gastrin causes proliferation of a variety of colorectal, gastric and pancreatic cancer cell lines, both in vitro and in vivo [11] [12] [6] [13] and that an autocrine pathway may be involved [14] [15]. Gly-G17 gastrin has also been shown to stimulate growth of various cancers via an autocrine/paracrine pathway [16] [17].
Gastrin is actually a family of peptides including G17 gastrin, G34 gastrin, and the immature forms, glycine-extended G17 (Gly-G17) gastrin and glycine-extended G34 (Gly-G34) gastrin. G17 and G34 share a 5-amino-acid carboxy-terminal sequence in common with cholecystokinin (CCK). It has been shown that in this sequence that interacts with the CCK-B/gastrin receptor [11] [18].
Gastrin requires post-translational carboxy-terminal alpha-amidation using glycine as the amide donor. The penultimate intermediate is gastrin with a C-terminal glycine—the so-called glycine-extended gastrins (Gly-G17 and Gly-G34). Similar concentrations of glycine-extended forms and mature gastrins are often found. Gly-G17 appears to have some of gastrin's biological activities [17].
Different tissues exhibit different patterns of post-translational gastrin modification resulting in the accumulation of different intermediates. While gastrin produced by the gastric antrum is largely fully amidated G17 [19], anterior pituitary corticotrophs almost entirely fail to process the amidation site, resulting in >99% glycine-extended gastrins. In neonatal pancreatic cells, gastrin is largely in the G34 form [19] and it is fully sulfated at Tyr29, a unique modification, making it CCK-like in its potency [3]. In neoplastic tissue, immature forms of gastrin typically predominate. In colorectal carcinomas, a considerable amount of progastrin species accumulates [20] [21]. In rat pancreatic tumor AR42J, it has been reported that only glycine-extended gastrins are present [17]. Nothing has been reported on the forms of gastrin produced by human pancreatic cancer cells.
The gastrin and CCK-B receptors were recently cloned and shown to be identical [22]. Messenger RNA for CCK-B receptors was detected in all pancreatic cancer cell lines of ductal origin and in normal pancreatic tissue as well as in fresh tumor cells [23] [24] [25] and may be over-expressed in malignant pancreatic tissue in comparison with normal tissue [26]. Some authors have detected CCK-B receptors using radiolabeled ligand binding (either gastrin or CCK) technologies in both normal pancreas and tumor cell lines [27] Others have failed to detect receptors in the tumor cell lines but do detect them in normal tissue [28] [29].
MacKenzie et al. [30] demonstrated by radioligand-binding the abundant presence of CCK-B/gastrin receptor on the rat pancreatic tumor cell line, AR42J. Tarasova et al. [31] have shown that following ligand binding assays, rapid clustering and internalization of the CCK-B/gastrin receptor occurred in the pancreatic tumor cell line AR42J, as well as in a variety of human gastric and colorectal tumor cell lines. Incubation of the CCK-B/gastrin receptor with 1 nM of the specific inhibitor CI-988 inhibited the proliferation of gastrin-stimulated (1 nM) AR42J cells by about 47% after 96 hours of treatment, which is consistent with competitive inhibition of the gastrin receptor [32]. Anti-G17 antibodies have been shown to inhibit the binding of gastrin to the CCK-B/gastrin receptors on the pancreatic tumor cell line AR42J.
It has been shown that gastrin peptides increase the proliferation of GI cancer cell lines of human and animal origin both in vitro and in vivo [5]. More recent studies with four human pancreatic cell lines have shown that all proliferation was increased by 40-68% G17 gastrin relative to untreated controls. Studies with receptor antagonists showed that this proliferative effect was mediated via the CCK-B receptor [11]. Other studies have reported similar results [12], but not all studies report a positive effect even if the presence of CCK-B receptors was confirmed by binding studies [25].
Additional studies compared the mitogenic effects of gastrin on colorectal and gastric tumor cells obtained from cancer subjects at surgical resection. It was shown that cells from 69% of gastric and 55% of colorectal tumors had an enhanced proliferation in response to G17 gastrin, which was of greater magnitude than that seen in normal cells obtained from the G1 mucosa [33] [34].
It has been shown that the gastrin gene is activated in epithelial cells derived from G1 tumor specimens, but not in normal G1 mucosal cells [35] [36] [37] [21] [17] [38]. Malignant epithelial cells have been shown to produce mitogenic gastrin peptides, which can increase self-proliferation of the surrounding cells, thereby inducing a state of tumor autonomy [39][16].
Gastrin also stimulated in vivo tumor growth in mice inoculated with human Panc-1 cells. Tumor volume in mice treated with pentagastrin was 127% greater than untreated control tumors, while those animals receiving a CCK-B receptor antagonist (without pentagastrin) had tumors only 60% as large as controls [11].
The stimulatory effect of gastrin was also demonstrated by antisense RNA directed at gastrin [40] which suppressed the growth of human pancreatic cell lines. The observation that gastrin mRNA is detectable in all normal as well as tumor cell lines and in fresh pancreatic tissue, while gastrin peptide is detectable only in malignant tissue, suggested that gastrin mRNA may be translated only in the tumor cells [15].
Although inactive in stimulating acid secretion, Gly-G17 gastrin has been shown to increase the proliferation of pancreatic [41] cancer cells, G17 and Gly-G17 were found to be equipotent in stimulating proliferation of rat pancreatic tumor AR42J cells.
The normal physiological functions of gastrin are mediated by CCK-B/gastrin receptors. Expression of the receptor occurs in all types of gastrointestinal malignancies including colorectal, gastric, pancreatic, hepatomas and colorectal liver metastases [42] [43][24] [15] [44] [45] [46]. Different isoforms of the receptor exist [51] [52], and more than one isoform of the CCK-B gastrin receptor may be co-expressed on individual cells. Therefore, antagonism of CCK-B receptors may not be the optimal method to suppress the proliferative action of gastrin present either in the serum or produced locally by the tumor cells.
It has been shown that several types of tumors, e.g., colorectal, stomach, pancreatic and hepatocellular adenocarcinomas, possess CCK-B/gastrin receptors in their plasma membranes and that they respond to gastrin with powerful cellular proliferation [53] [13]. Furthermore, more recently it has been discovered that many of these cancer cells also secrete gastrin and thus effect an autonomous proliferative pathway [21] [37] [16].
The CCK-B/gastrin receptor belongs to a family of G protein-coupled receptors with seven transmembrane domains with equal affinity for both CCK and gastrin [54]. This receptor was named a CCK type-B receptor because it was found predominantly in the brain [55]. The receptor was subsequently found to be identical to the peripheral CCK/gastrin receptor in the parietal and ECL cells of the stomach [56]. This receptor has been well characterized in a number of normal [57] [58] and tumor tissues [59] [34], and extensively studied using the rat pancreatic adenocarcinoma cell line AR42J [60]. The AR42J CCK-B/gastrin receptor cDNA has been cloned and sequenced, and it is more than 90% homologous in DNA sequence to the CCK-B/gastrin receptor in rat and human brain, and more than 84% homologous in sequence to the canine parietal cell CCK-B/gastrin receptor cDNA [61], demonstrating a high sequence homology even between species.
The peptide hormones G117 and G34 bind to the CCK-B/gastrin receptor on the cell membrane of normal cells. However, it has been found that G17, and not G34, stimulates the growth of gastrin-dependent cancer cells. Serum-associated G17, in particular, has the potential to stimulate the growth of colorectal tumors in an endocrine manner mediated by CCK-B/gastrin receptors [34] in the tumor cells. Gastrin-17 appears to be particularly implicated in stimulating the growth of colorectal adenocarcinomas due to a possible increased affinity for the CCK-B/gastrin receptor on the tumor cells, over other gastrin hormone species [62]. The CCK-B/gastrin receptors were found to be expressed in a high affinity form on 56.7% of human primary colorectal tumors [53]. It has been postulated that a potential autocrine loop may also exist due to endogenous production of precursor gastrin peptides by such tumors [21]. The resulting G17 ligand/receptor complex stimulates cell growth by way of secondary messengers for regulating cell function [63]. The binding of G17 to the CCK-B/gastrin receptor leads to activation of phosphatidyl inositol breakdown, protein kinase C activation with a resultant increase in intracellular calcium ion concentration, as well as the induction of c-fos and c-jun genes via mitogen-activated protein kinase, which has been implicated in the regulation of cell proliferation [64]. Additionally, gastrin binding to the CCK-B/gastrin receptor has been associated with the subsequent increase in phosphorylation by a tyrosine kinase, pp125FADK (focal adhesion kinase), which may also have a role in the transmission of mitogenic signals [65].
A number of high affinity CCK-B/gastrin receptor antagonists have been evaluated therapeutically both in vitro and in vivo in a number of experimental models of gastrointestinal cancer. For example, proglumide, a glutamic acid derivative [16] [66] [67] Benzotript, an N-acyl derivative of tryptophan; L-365,260, a derivative of Aspercillin [68], and CI-988 a molecule that mimics the C-terminal pentapeptide sequence of CCK [69] have been shown to effectively neutralize the effects of exogenous gastrin on gastrointestinal tumor growth both in vitro and in vivo [6] [70]. However, these antagonists have severe toxic side effects and lack specificity as they block the action of all potential ligands of the receptor such as G34 and CCK in normal cells. Recently, highly potent and selective CCKB/gastrin receptor antagonists such as YM022 [71] and YF476 [72] have been also described.
Proglumide and Benzotript have been widely assessed in pre-clinical studies. The main problem with these compounds is their lack of potency, with relatively high concentrations required to displace G17. Despite this, proglumide and benzotript inhibited the basal and gastrin-stimulated proliferation of a number of cell lines [67]. In addition, proglumide increased the survival of xenograft mice bearing the gastrin-sensitive mouse colon tumor, MC26 to 39 days in the treated animals from 25 days in the control animals.
Due to the low specificity of this class of gastrin antagonizing agents for the gastrin/CCKB receptor, the inhibition of tumor growth may not be effectively control with gastrin antagonists. Moreover, the cellular receptors which recognize and bind the gastrins do not bind all the inhibitors tested [16]. Thus, if complete inhibition of gastrin binding to the receptor does not occur in the autocrine growth cascade, then the gastrin antagonists may be unable to block this mechanism of tumor growth promotion.
Recent developments have demonstrated the feasibility of immunoneutralization of hormones or their receptor moieties in order to inhibit the hormone controlled physiological functions or effects, such as cellular growth. (U.S. Pat. Nos. 5,023,077 and 5,468,494)
For example, immunization with the immunogen G17DT elicits antibodies that react specifically with the aminoterminal end of G17 gastrin and Gly-G17 gastrin (U.S. patent application Ser. No. 08/798,423). The antibodies do not cross-react with any of the other gastrin species tested, including G34 gastrin and CCK. Antibodies elicited by G17DT inhibit the binding of gastrin to the CCK-B/gastrin receptor on a variety of gastrointestinal tumor cells, including pancreatic tumor cells. Antibodies elicited by G17DT inhibit the growth of human gastric, pancreatic, and colorectal cancer cells in vitro and in in vivo animal models of gastric and colorectal cancer. Immunological neutralization has been discovered to inhibit metastsis of colorectal cancer [46] [47].
The alternate or additional immunological weapon against the gastrin effect on pancreatic cancer growth comprises the induction of anti-CCKB/gastrin receptor antibody binding with a specific anti-receptor GRE1 or GRE4 peptide epitope, as described in co-assigned pending U.S. patent application Ser. No. 09/076,372. Accordingly, the receptor moieties can be prevented from binding the circulating gastrin hormone or fragments thereof. Furthermore, this immunological inhibition of pancreatic cancer advantageously results in the internalization of the receptor antibody complex causing apoptosis-like cell death.
Certain anticancer chemical compounds have been found useful for treating adenocarcinoma such as pancreatic tumors. For example, Gemcitabine (2′, 2′, difluorodeoxycytidine) is a nucleoside analog with structural similarities to cytarabine. Its mode of action involves disruption of cell replication. Gemcitabine enters the cell via a carrier-mediated transport system that is shared with other nucleosides. It is phosphorylated sequentially to difluorodeoxycytidine monophosphate (diFdCMP), difluorodeoxycytidine diphosphate (diFdCDP) and difluorodeoxycytidine triphosphate (diFdCTP). Preclinical studies of gemcitabine have shown incorporation of the phosphorylated diFdCTP into DNA [73][74].
Gemcitabine triphosphate is a substrate and competitive inhibitor of DNA polymerases alpha and epsilon. Once dFdCTP is incorporated into the growing chain, only one (or perhaps two) more nucleotide(s) can be incorporated, a novel mechanism termed “masked chain termination.” Once additional residues are incorporated at the 3′ end, gemcitabine cannot be excised by the proofreading exonucleolytic activity of DNA polymerase [48]. DNA fragmentation and apoptosis follow. As predicted by its mode of action, gemcitabine is active only in S-phase when cells are actively replicating DNA [49].
Since pancreatic cancer has a high occurrence of metastasis, this method also comprises advantageous combination treatment with immunological anti-gastrin, anti-CCK-B/gastin receptor agents and chemotherapeutic agents such as irinotecan and optionally 5-FU/LV or gemcitabine, or both.
Irinotecan is a chemotherapeutic drug (CAMPOSAR), which has been approved for some types of cancer, mostly as second-live treatment. It has been applied in conjunction with 5-FU/LV against metastatic colorectal carcinoma which progressed after 5-FU treatment.
Cisplatin is a drug used in a variety of neoplasms that is capable of producing inter- and intrastrand DNA cross-links. Cisplatin can be administered alone or together with other chamotherapeucics.
In view of the very poor prognosis of pancreatic cancer and lack of significant survival afforded by the currently available therapies, a therapeutic strategy involving immunological targeting of gastrin and its receptor in combination with chemotherapeutic methods using one or more chemotherapeutic agents may provide a novel and efficacious therapy.